Changes In Cytosolic Calcium Research Articles

Effects of Anandamide and Noxious Heat on Intracellular Calcium Concentration in Nociceptive DRG Neurons of Rats

As an endogenous agonist at the cannabinoid receptor CB1 and the capsaicin-receptor TRPV1, anandamide may exert both anti- and pronociceptive actions. Therefore we studied the effects of anandamide and other activators of both receptors on changes in free cytosolic calcium ([Ca(2+)](i)) in acutely dissociated small dorsal root ganglion neurons (diameter: < or =30 microm). Anandamide (10 microM) increased [Ca(2+)](i) in 76% of the neurons. The EC(50) was 7.41 microM, the Hill slope was 2.15 +/- 0.43 (mean +/- SE). This increase was blocked by the competitive TRPV1-antagonist capsazepine (10 microM) and in Ca(2+)-free extracellular solution. Neither exclusion of voltage-gated sodium channels nor additional blockade of voltage-gated calcium channels of the L-, N-, and/or T-type, significantly reduced the anandamide-induced [Ca(2+)](i) increase or capsaicin-induced [Ca(2+)](i) transients (0.2 microM). The CB1-agonist HU210 (10 microM) inhibited the anandamide-induced rise in [Ca(2+)](i). Conversely, the CB1-antagonist AM251 (3 microM) induced a leftward shift of the concentration-response relationship by approximately 4 microM (P < 0.001; Hill slope, 2.17 +/- 0.75). Intracellular calcium transients in response to noxious heat (47 degrees C for 10 s) were highly correlated with the anandamide-induced [Ca(2+)](i) increases (r = 0.84, P < 0.001). Heat-induced [Ca(2+)](i) transients were facilitated by preincubation with subthreshold concentrations of anandamide (3 microM), an effect that was further enhanced by 3 microM AM251. Although anandamide acts on both TRPV1 and CB1 receptors in the same nociceptive DRG neurons, its pronociceptive effects dominate. Anandamide triggers an influx of calcium through TRPV1 but no intracellular store depletion. It facilitates the heat responsiveness of TRPV1 in a calcium-independent manner. These effects of anandamide differ from those of the classical exogenous TRPV1-agonist capsaicin and suggest a primarily modulatory mode of action of anandamide.

When Evidence Is Scant, Mathematical Modeling Offers a Roadmap for Discovery

When Evidence Is Scant, Mathematical Modeling Offers a Roadmap for Discovery

Fatty Acid Ethyl Ester Effects on Interleukin‐2 Production, Cyclic AMP Synthesis, and Calcium Influx in Human Mononuclear Cells

Fatty acid ethyl esters (FAEE), nonoxidative ethanol metabolites, are produced by the esterification of fatty acids and ethanol. Plasma and serum FAEE correlate linearly with blood ethanol levels and are present in organs most commonly damaged by ethanol abuse. Our previous studies have shown that there is significant synthesis of FAEE by human mononuclear cells within seconds of exposure to physiologic doses of ethanol. We studied the effects of FAEE on selected early events of mononuclear cell activation that follow stimulation with phytohemagglutinin (PHA). Fatty acid ethyl esters induced changes in cytosolic free calcium ([Ca2+]) levels, the production and secretion of interleukin-2 (IL-2) by the cells, and intracellular cAMP concentrations. A mononuclear fraction of human white blood cells (WBC) was incubated with physiologic doses of FAEE. For experiments involving IL-2 production and calcium influx, PHA-stimulated cells were incubated with 10, 25, 50, or 100 microM ethyl oleate, a representative FAEE species, for 60 minutes. Interleukin-2 was measured by enzyme immunometeric assay and maximum levels of Ca2+ were monitored by spectrofluorimetry. In other experiments, mononuclear cells were incubated with 10, 25, and 50 microM ethyl oleate for 0.08 to 120 minutes, and then the concentration of cAMP was determined by a cAMP competitive enzyme immunoassay system. Fatty acid ethyl esters inhibited the PHA-induced IL-2 production and secretion in activated human mononuclear cells. Fatty acid ethyl esters also inhibited PHA-induced [Ca2+] influx into cells in a dose-dependent fashion. There was a rapid increase in the intracellular cAMP concentration of mononuclear cells induced by FAEE, with FAEE dose dependence. The cAMP concentration decreased as the incubation time with FAEE was increased. Fatty acid ethyl esters inhibited PHA-stimulated IL-2 production and Ca2+ influx into human mononuclear cells and elevated intracellular cAMP concentration. These changes in mononuclear cell signaling may be associated with the immunosuppression associated with alcohol abuse.

Thrombin-induced caspases 3 and 9 translocation to the cytoskeleton is independent of changes in cytosolic calcium in human platelets

Apoptosis has been shown to be associated with changes in cytosolic free calcium concentration ([Ca 2+] c). Here we show that the agonist thrombin induces activation of caspases 9 and 3 and translocation of the caspase active forms and procaspases to the cytoskeleton in human platelets. Dimethyl-BAPTA loading did not affect thrombin-induced caspase 9 and 3 activation or translocation suggesting that these responses are independent of increases in [Ca 2+] c. Treatment with thapsigargin plus ionomycin, to induce extensive Ca 2+ store depletion and subsequent increase in [Ca 2+] c, stimulates caspase activation although it was unable to induce caspase translocation to the cytoskeleton. Similar results were observed in cells loaded with dimethyl-BAPTA, suggesting that activation of caspases 9 and 3 by thapsigargin plus ionomycin does not require rises in [Ca 2+] c. These findings suggest that thrombin-induced caspase 9 and 3 activation and translocation are independent on rises in [Ca 2+] c but might require store depletion in human platelets.

Serotonin-Induced Inhibition of K V Current

See related article, pages 931–938 Pulmonary vascular tone is largely determined by potassium and calcium currents in the smooth muscle cells (SMCs) of the small resistance arteries. Under normoxic circumstances an outward potassium current ( I K) passes through voltage-gated (Kv), calcium-activated (KCa), and background tandem-pore domain (K2P) potassium channels. The latter (coded by the KCNK family of genes) include the twik-related acid sensitive K+ channel TASK and TASK-like channels. This current keeps the resting membrane potential in the range of −50 to −60 mV and inhibits calcium entry through L-type calcium channels.1 Vasodilator substances such as nitric oxide and prostacyclin, released from the endothelium, increase I K, one of several actions that lead to further vasodilatation.2,3 Vasoconstrictor substances, such as endothelin, can inhibit I K4 and/or release calcium from the sarcoplasmic reticulum.5 In addition to vasoactive effects, an increase in cytosolic potassium inhibits smooth muscle cell apoptosis,6 and an increase in cytosolic calcium promotes cellular proliferation.7 Consequently, the inhibition of I K is important not only in causing membrane depolarization and calcium entry but also in stimulating vascular remodeling and in the development of chronic pulmonary hypertension. Agents that can cause pulmonary hypertension, such as the anorectic drugs and hypoxia, inhibit I K8,9 and also enhance calcium release from the sarcoplasmic reticulum, leading to subsequent repletion of calcium through store-operated channels.10,11,12 The smooth muscle cells in the resistance pulmonary arteries of patients with pulmonary arterial hypertension (PAH) exhibit both reduced I K and decreased expression of Kv channels,13 and also increased expression of the TRPC ( t ransient r eceptor p otential, c anonical) genes that code for store-operated and receptor-operated calcium channels.14 The decreased expression and function of the Kv channels may …

Loss of Polycystin-1 in Human Cyst-Lining Epithelia Leads to Ciliary Dysfunction

A "two-hit" hypothesis predicts a second somatic hit, in addition to the germline mutation, as a prerequisite to cystogenesis and has been proposed to explain the focal nature for renal cyst formation in autosomal dominant polycystic kidney disease (ADPKD). It was reported previously that Pkd1(null/null) mouse kidney epithelial cells are unresponsive to flow stimulation. This report shows that Pkd1(+/null) cells are capable of responding to mechanical flow stimulation by changing their intracellular calcium concentration in a manner similar to that of wild-type cells. This paper reports that human renal epithelia require a higher level of shear stress to evoke a cytosolic calcium increase than do mouse renal epithelia. Both immortalized and primary cultured renal epithelial cells that originate from normal and nondilated ADPKD human kidney tubules display normal ciliary expression of the polycystins and respond to fluid-flow shear stress with the typical change in cytosolic calcium. In contrast, immortalized and primary cultured cyst-lining epithelial cells from ADPKD patients with mutations in PKD1 or with abnormal ciliary expression of polycystin-1 or -2 were not responsive to fluid shear stress. These data support a two-hit hypothesis as a mechanism of cystogenesis. This report proposes that calcium response to fluid-flow shear stress can be used as a readout of polycystin function and that loss of mechanosensation in the renal tubular epithelia is a feature of PKD cysts.

Cyst‐lining epithelial cells from ADPKD kidneys have a mechano‐ciliary dysfunction

Background Studies have shown that cilia are mechanical fluid shear sensors. We have reported previously that polycystins, ADPKD proteins, play a role in mechanosensation in mouse kidney epithelial cilia. Objective The current study was designed to analyze the functions of polycystins in human cells. Methods Epithelial cells were isolated from normal and ADPKD (both normal-looking and dilated/cystic tubules) kidneys. These cells were subject to fluid shear analysis. Results Both immortalized and primary cultured renal tubular cells originating from normal human and non-dilated ADPKD patient kidneys displayed normal ciliary expressions of the polycystins and responded to fluid flow shear stress with a change in cytosolic calcium. In contrast, immortalized and primary cultured cyst-lining epithelial cells from ADPKD patients with PKD1 mutation or with abnormal ciliary expression of polycystins were not responsive to fluid shear stress. Conclusion Cyst-lining epithelia in ADPKD kidneys have lost their ciliary mechanosensing ability. Thus, we propose that loss of mechanosensation in the renal tubular epithelia is a feature of the cyst lining epithelia and monitoring cytosolic calcium in this flow assay is a useful functional readout of polycystin function. This work is supported by the NIH and PKD foundation.

Aldosterone Regulation of the Cardiac Sodium‐Calcium Exchanger

The objective of this study was to determine whether the protein expression and functional activity of the cardiac sodium-calcium exchanger was subject to regulation by activation of mineralocorticoid receptors. The studies were carried out in cultured adult rat ventricular myocytes. Protein expression was evaluated by Western blotting, using a monoclonal antibody to the NCX1 protein (R3F1, Swant, Switzerland). Functional activity of the sodium-calcium exchanger was evaluated by first inducing reverse-mode exchanger activity and then using fluorescent calcium indicators to measure changes in cytosolic calcium. Aldosterone strongly inhibited exchanger protein expression in a dose-dependent manner (0.1–10 nM). This effect depended on mineralocorticoid activation, as it could be blocked by spironolactone. Cytosolic calcium measurements confirmed that the decreased protein expression seen with 10 nM aldosterone was accompanied by a significant inhibition of sodium-calcium exchanger functional activity. This work was supported by NHLBI R01HL056910 (RWH).

Dopamine D2-like Receptors Are Expressed in Pancreatic Beta Cells and Mediate Inhibition of Insulin Secretion

Dopamine signaling is mediated by five cloned receptors, grouped into D1-like (D1 and D5) and D2-like (D2, D3 and D4) families. We identified by reverse transcription-PCR the presence of dopamine receptors from both families in INS-1E insulin-secreting cells as well as in rodent and human isolated islets. D2 receptor expression was confirmed by immunodetection revealing localization on insulin secretory granules of INS-1E and primary rodent and human beta cells. We then tested potential effects mediated by the identified receptors on beta cell function. Dopamine (10 microM) and the D2-like receptor agonist quinpirole (5 microM) inhibited glucose-stimulated insulin secretion tested in several models, i.e. INS-1E beta cells, fluorescence-activated cell-sorted primary rat beta cells, and pancreatic islets of rat, mouse, and human origin. Insulin exocytosis is controlled by metabolism coupled to cytosolic calcium changes. Measurements of glucose-induced mitochondrial hyperpolarization and ATP generation showed that dopamine and D2-like agonists did not inhibit glucose metabolism. On the other hand, dopamine decreased cell membrane depolarization as well as cytosolic calcium increases evoked by glucose stimulation in INS-1E beta cells. These results show for the first time that dopamine receptors are expressed in pancreatic beta cells. Dopamine inhibited glucose-stimulated insulin secretion, an effect that could be ascribed to D2-like receptors. Regarding the molecular mechanisms implicated in dopamine-mediated inhibition of insulin release, our results point to distal steps in metabolism-secretion coupling. Thus, the role played by dopamine in glucose homeostasis might involve dopamine receptors, expressed in pancreatic beta cells, modulating insulin release.

A gibberellin-regulated calcineurin B in rice localizes to the tonoplast and is implicated in vacuole function.

Many developmental and environmental signals are transduced through changes in intracellular calcium concentrations, yet only a few calcium-binding proteins have been identified in plants. Calcineurin B-like (CBL) proteins are calcium-binding proteins that are thought to function as plant signal transduction elements. RNA profiling using a rice (Oryza sativa cv Nipponbare) oligonucleotide microarray was used to monitor gene expression in de-embryonated rice grains. This analysis showed that a putative rice CBL gene responded to gibberellic acid, but not abscisic acid, treatment. The CBL gene family in rice contains at least 10 genes and these have extensive similarity to the CBLs of Arabidopsis (Arabidopsis thaliana). In yeast (Saccharomyces cerevisiae) two-hybrid assays, rice CBLs interact with the kinase partners of Arabidopsis CBLs. Only one rice CBL gene, OsCBL2, is up-regulated by GA in the aleurone layer. A homolog with 91% sequence identity to OsCBL2 was cloned from barley (Hordeum vulgare cv Himalaya), and designated HvCBL2. We examined the localization and function of OsCBL2 and HvCBL2 in rice and barley aleurone because changes in cytosolic calcium have been implicated in the response of the aleurone cell to GA. Green fluorescent protein translational fusions of OsCBL2 and OsCBL3 were localized to the tonoplast of aleurone cell protein storage vacuoles and OsCBL4-green fluorescent protein was localized to the plasma membrane. Data from experiments using antisense expression of OsCBL2 and HvCBL2 are consistent with a role for OsCBL2 in promoting vacuolation of barley aleurone cells following treatment with GA.

Effect of 17??-Estradiol on Insulin Secretion and Cytosolic Calcium in Min6 Mouse Insulinoma Cells and Human Islets of Langerhans

Female gonadal steroids can exert an insulinotropic effect in vivo. The objective of this study was to investigate the effects in vitro of 17-beta-estradiol (17beta-E2) on changes in cytosolic calcium ([Ca]i) and on insulin secretion from the MIN6 mouse insulinoma cell line and human primary islets of Langerhans. Stimulus-induced changes in [Ca]i were measured in Fura-2-loaded cells by single cell microfluorimetry. The effects of 17beta-E2 on insulin secretion were measured in static incubation experiments, and the rate and pattern of secretory responses were studied in multi-channel perifusion experiments. 17Beta-E2 (1-100 nmol/L) enhanced basal (2 mmol/L glucose) insulin secretion but had no effect on secretory responses to 20 mmol/L glucose or to depolarizing stimuli (100 micromol/L tolbutamide, 20 mmol/L KCl). Approximately 60% of MIN6 cells responded to 17beta-E2 (1-100 nmol/L) with a small but sustained increase in [Ca]i, whereas 98% of MIN6 cells responded to tolbutamide (100 micromol/L). Similar effects were observed in experiments using human primary beta cells. In contrast, 17beta-E2 had no detectable effect on the increases in [Ca]i evoked by tolbutamide (100 micromol/L) or glucose (20 mmol/L). Our observations are consistent with a rapid effect of 17beta-E2 to depolarize beta cells leading to an influx of extracellular Ca and the initiation of insulin secretion by the consequent elevations in [Ca]i. We suggest that this may offer a mechanism through which circulating estradiol can influence beta-cell responsiveness to other signals.

Interleukin-8 primes oxidative burst in neutrophil-like HL-60 through changes in cytosolic calcium

In response to a variety of stimuli, neutrophils release large amount of reactive oxygen species (ROS) generated by NADPH oxidase. This process known as the respiratory burst is dependent on cytosolic free calcium concentration ([Ca 2+] i). Proinflammatory cytokines such as interleukin-8 (IL-8) may modulate ROS generation through a priming phenomenon. The aim of this study was to determine the effect of human IL-8 on ROS production in neutrophil-like dimethylsulfoxide-differentiated HL-60 cells (≠HL-60 cells) and further to examine the role of Ca 2+ mobilization during the priming. IL-8 at 10 nM induced no ROS production but a [Ca 2+] i rise (254 ± 36 nM). IL-8 induced a strongly enhanced (2 fold) ROS release during stimulation with 1 μM of N-formyl- l-methionyl- l-leucyl- l-phenylalanine (fMLF). This potentiation of ROS production is dependent of extracellular Ca 2+ (17.0 ± 4.5 arbitrary units (A.U.) in the absence of Ca 2+ versus 56.6 ± 3.9 A.U. in the presence of 1.25 mM of Ca 2+). Also, IL-8 enhanced fMLF-stimulated increase in [Ca 2+] i (375 ± 35 versus 245 ± 21 nM, 0.1 μM of fMLF). IL-8 had no effect on ≠HL-60 cells in response to 1 μM of thapsigargin (472 ± 66 versus 470 ± 60 nM). In conclusion, Ca 2+ influx is necessary for a full induction of neutrophil priming by IL-8.

Similarities of K+ATP Channel Expression and Ca2+ Changes in Pancreatic ?? Cells and Hypothalamic Neurons

The mechanisms through which pancreatic beta cells recognize and respond to changes in circulating glucose are well understood. Evidence is accumulating that a subpopulation of neurons in the ventromedial hypothalamus (VMH) use similar cellular mechanisms to sense changes in extracellular glucose. In the present study, we used PCR and single-cell calcium imaging techniques to investigate whether glucose-sensing cells in the pancreas and hypothalamus employ a similar set of stimulus-response elements. Dispersed cells from mouse pancreata and hypothalamus were used in conjunction with the insulin-secreting cell line MIN6. We present functional data suggesting that both pancreatic and a subpopulation of hypothalamic cells exhibit glucose- and tolbutamide-evoked changes in cytosolic calcium and consider some clinical implications of different glucose sensors using the same mechanisms.

Electrical synapse formation disrupts calcium‐dependent exocytosis, but not vesicle mobilization

Electrical coupling exists prior to the onset of chemical connectivity at many developing and regenerating synapses. At cholinergic synapses in vitro, trophic factors facilitated the formation of electrical synapses and interfered with functional neurotransmitter release in response to photolytic elevations of intracellular calcium. In contrast, neurons lacking trophic factor induction and electrical coupling possessed flash-evoked transmitter release. Changes in cytosolic calcium and postsynaptic responsiveness to acetylcholine were not affected by electrical coupling. These data indicate that transient electrical synapse formation delayed chemical synaptic transmission by imposing a functional block between the accumulation of presynaptic calcium and synchronized, vesicular release. Despite the inability to release neurotransmitter, neurons that had possessed strong electrical coupling recruited secretory vesicles to sites of synaptic contact. These results suggest that the mechanism by which neurotransmission is disrupted during electrical synapse formation is downstream of both calcium influx and synaptic vesicle mobilization. Therefore, electrical synaptogenesis may inhibit synaptic vesicles from acquiring a readily releasable state. We hypothesize that gap junctions might negatively interact with exocytotic processes, thereby diminishing chemical neurotransmission.

Mechanisms of Interleukin-4 Effects on Calcium Signaling in Airway Smooth Muscle Cells

In airway smooth muscle cells, interleukin (IL)-4 inhibited both carbachol- and caffeine-induced calcium mobilization from the sarcoplasmic reticulum (SR). Because of the known signaling pathways for IL-4 and importance of calcium uptake in maintaining SR calcium stores shared by agonists and caffeine, it was hypothesized that this rapid inhibitory effect might depend on phosphatidylinositol 3-kinase (PI3K) and on inhibition of calcium uptake by the SR. Enzyme-dispersed bovine trachealis cells were loaded with Fura-2/acetoxymethyl ester, and changes in cytosolic calcium were imaged in single cells. Cells were pretreated with inhibitors of PI3K, either wortmannin (100 nM), LY294002 [2-(4-morpholinyl)-8-phenyl-4H-1-benzopyran-4-one] (50 microM), or deguelin (100 nM). Calcium transients in response to carbachol (10 microM) were significantly decreased to 0.34 +/- 0.10 of control after 20-min treatment with IL-4 but were 1.10 +/- 0.26 and 1.08 +/- 0.23 when wortmannin or deguelin, respectively, was added along with IL-4. LY294002 alone had nonspecific effects on transients. In other experiments, cyclopiazonic acid (CPA) (5 microM), an inhibitor of SR calcium uptake, decreased carbachol-stimulated transients within 4 min to 0.83 +/- 0.08 of control (n = 6). However, for cells treated with IL-4 (50 ng/ml) plus CPA, transients decreased significantly more, to only 0.51 +/- 0.05 (n = 6; p < 0.05). Longer exposures to IL-4 and a higher concentration of CPA (30 microM) gave similar results. It was concluded that IL-4 did not inhibit transients in the presence of PI3K antagonists but that it did in the presence of CPA. This suggested that IL-4 inhibited calcium transients by mechanisms dependent upon a wortmannin-sensitive PI3K but not by inhibition of calcium uptake into the SR.

Elevated Ca 2+i transients induced by trimethyltin chloride in HeLa cells: types and levels of response

Humans are exposed to organotins, like trimethyltin (TMT) chloride via air, water and food, and intoxication might result in severe health complications. Toxic effects of organotin compounds are well documented, but possible mechanisms remain unclear and only little information is available how organometallic species interact with calcium controlling mechanisms. Therefore, the aim of this work was to investigate the effects of TMT on calcium homeostasis in HeLa S3 cells. Dynamic changes of cytosolic calcium (Ca 2+ i) were monitored using laser-scanning microscopy and fluo-4 loaded cells. Application of TMT resulted in sustained as well as in transient elevations of Ca 2+ i. The number of reacting cells was directly correlated to the concentration of TMT used: with 500 μM TMT all cells reacted, with 50 μM TMT 80% and with 5 μM 74%. The fast Ca 2+ i-transients (spikes), measured in single cells, occurred even with 0.25 μM TMT and varied in size and duration. The sustained increase of Ca 2+ i, measured as the average over all cells, was dose dependent with an ∼8% increase for 5 μM TMT, ∼12.3% for 50 μM and ∼145% for 500 μM TMT. Moreover, this effect was partly reversible. A second application resulted in a similar sustained rise of Ca 2+ i compared to the first application of TMT, there was also no difference when no calcium was added to the external solution (151 ± 10% compared to 145 ± 15%; 500 μM TMT). This rise of Ca 2+ i was highly reduced (<10% increase) when the internal calcium stores were depleted before TMT (500 μM) was applied. Our data suggest that TMT influences Ca 2+ i-homeostasis of HeLa S3 cells, which might be related to its toxicity in this cell line.

Effects of Protein Tyrosine Kinase, Protein Tyrosine Phosphatase and Protein Kinase C on the Apoptotic Process of Leukemia Cells and Human Cortex Neurons Managed by Arsenic Trioxide.

Objective Study on the effects of protein tyrosine kinase, protein tyrosine phosphatase and protein kinase C (PKC)on apoptotic process, observe the changes of cytosolic calcium ([Ca2+]i) of human leukemia cells and cortex neurons after clinical relevant dosage of arsenic trioxide (As2O3) exposure in vitro.Methods Marked [Ca2+]I of human leukemia cells and cortex neurons with Fluo-3/AM, assayed the changes of [Ca2+]I with laser confocal microscopy in real-time after managed with As2O3 in different concentrations respectively, detected the effects of protein tyrosine kinase, the protein tyrosine phosphatase and the activation of protein kinase C on these changes with confocal microscopy and Phosphorus radioisotope assay. Assayed the DNA ladders of leukemia cells and cortex neurons after exposed in As2O3.Results As2O3 1μmol/L can increase the [Ca2+]I of NB4 cells remarkably but show no effects on neurons. Vanadate, a kind of protein tyrosine phosphatase inhibitor, can dose-dependently promote the increasing of [Ca2+]I caused by 2, 5, 10μmol/L As2O3.The mean value of total increased rate in 280 seconds after exposed in As2O3 in different concentrations were: 6.5±2.3%, 21.7±2.1 %,49.2±2.5% in NB4 cells, and 6.7±2.1%, 19.4±2.5 %, 52.3±2.7% in cortex neurons respectively. Genistein, a kind of protein tyrosine kinase inhibitor, can dose-dependently decrease the raising of [Ca2+]I caused by 2, 5, 10μmol/L As2O3.The mean value of total inhibited rate in 280 seconds after As2O3 management in different concentrations were 6.7±2.9%, 25.6±2.5 %, 52.2±3.5% in NB4 cells, and 7.8±3.1%, 18.1±2.8%, 51.3±3.3% in cortex neurons respectively. The activation of PKC began when exposed in As2O31μmol/L for 3 h, and keep raising continuously in NB4 cells., DNA ladders appeared after exposed in As2O31μmol/L for 24h. However, no appearances above were found in human cortex neurons. In 2μmol/L As2O3 exposedness, The activation of PKC and DNA ladders appeared in neurons.Conclusions The phosphorylation and dephosphorylation of protein tyrosine kinase and protein tyrosine phosphatase participated in nonspecific apoptosis signal transduction pathway of As2O3, and accompanied with PKC activation. The [Ca2+] elevation was closely related to PKC increasing activation. The tolerances between leukemia cell and cortex neuron to As2O3 were differ in dosage.

Immunolocalization of type 2 inositol 1,4,5-trisphosphate receptors in cardiac myocytes from newborn mice.

The precise localization and role of inositol 1,4,5-trisphosphate (InsP(3)) receptors (InsP(3)Rs) in cardiac muscle cells are largely unknown. It is believed that waves and oscillations in cytosolic free calcium triggered by activation of InsP(3)Rs underlie modifications of cellular responses that lead to changes in gene expression in other cells. However, how changes in cytosolic calcium alter gene expression in cardiac cells is unknown. Moreover, it is unclear how changes in cytosolic calcium that alter gene expression do so independently of effects of calcium on other cellular functions, such as contraction. Here we show that InsP(3)R type 2 is the only isoform present in cardiac myocytes isolated from neonatal mouse ventricles. We also show that type 2 InsP(3)Rs are associated with the nucleus and that activation of type 2 InsP(3)Rs with endothelin-1 or phenylephrine selectively increases transcription of atrial natriuretic factor and skeletal alpha-actin. Type 2 InsP(3)Rs are also in striations. Activation of InsP(3)Rs with adenophostin A in permeabilized cells induced calcium release in the nuclear domain and other regions of the cell away from the nucleus. Agonist-induced increase in gene expression and calcium release were blocked by the InsP(3)R inhibitors 2-aminoethoxydiphenyl borate and xestospongin C. The spatial separation of type 2 InsP(3)Rs provides support for the concept that microdomains of calcium discretely alter various cell processes. Our experiments suggest that calcium released by InsP(3)Rs in the nuclear domain provides a direct mechanism for the control of gene expression, whereas release of calcium in the cytoplasm may modulate other processes, such as contraction.

Cooperative activation of cultured vagal afferent neurons by leptin and cholecystokinin.

To test the hypothesis that leptin can directly activate vagal afferent neurons, we used fluorescence imaging to detect acute changes in cytosolic calcium after leptin application to primary cultures of vagal afferent neurons dissociated from adult rat nodose ganglia. We found that approximately 40% of vagal afferent neurons exposed to leptin (40 ng/ml) responded with rapid and reversible increases in cytosolic calcium. These responses were dependent upon extracellular calcium. As previously reported, about 35% of vagal afferents increase cytosolic calcium in response to the gut-peptide cholecystokinin (CCK). A majority (74%) of neurons that responded to CCK also exhibited increases in cytosolic calcium in response to leptin. In addition, synergistic increases in cytosolic calcium were observed when leptin and CCK were applied in combination. These results demonstrate that leptin acts directly on vagal afferent neurons to trigger acute influxes of extracellular calcium. Our results also suggest cooperation between leptin and CCK in the activation of some vagal afferent neurons. Acute activation of vagal afferents by leptin alone and in combination with CCK may contribute to modulation of visceral reflexes and control of food intake.

Adhesion and signaling by B cell-derived exosomes: the role of integrins.

Exosomes are nanometer-sized vesicles secreted by various cells, with potentially diverse roles in physiology. Although emphasis has been placed on their involvement in immune modulation, their potential for more wide-ranging biological effects has not been appreciated. A common exosome feature is the expression of adhesion molecules, which include the integrin family. We have for the first time addressed the possible function of B cell-derived exosome-integrins by examining adhesive interactions of exosomes (immobilized onto beads) with extracellular matrix (ECM) components and cytokine-treated fibroblasts. Integrin (beta1 and beta2) expression was demonstrated by Western blotting and flow cytometry. Binding studies (with blocking antibodies) demonstrated their function in adhesion to collagen-I, fibronectin, and tumor necrosis factor (TNF)-alpha-activated fibroblasts. Exosome adhesion to TNF-alpha-activated fibroblasts also triggered integrin-dependent changes in cytosolic calcium, measured by single cell imaging. Thus, B cell-derived exosomes express functional integrins, which are capable of mediating anchorage to ECM and cell-surface adhesion molecules, and may be a novel mode of delivering adhesion signals at distances beyond that of direct cell-cell contact during inflammation.